1. Field of the Invention
This invention relates to radiodiagnostic agents and reagents for preparing such agents, and also methods for producing radiolabeled radiodiagnostic agents. Specifically, the invention relates to technetium-99m (.sup.99m Tc) labeled agents, methods and kits for making the agents, and methods for using the agents to image pathological sites, including sites of infection, inflammation, cancer and atherosclerosis in a mammalian body. Specifically the agents and reagents are derivatives of oligosaccharides, more specifically .beta.-glucans.
2. Description of the Prior Art
In the field of nuclear medicine, certain pathological conditions can be localized or the extent of such conditions determined by imaging the internal distribution of administered radioactively-labeled tracer compounds (i.e. radiotracers or radiopharmaceuticals) that accumulate specifically at the pathological site. This type of procedure is commonly known as radioimaging or scintigraphic imaging. Radioimaging has particular advantages over other methods of diagnosis in that it is essentially non-invasive, highly sensitive, highly specific, can be used to scan the entire body and is relatively cost-effective. A variety of radionuclides are known to be useful for radioimaging, including .sup.67 Ga, .sup.68 Ga, .sup.99m Tc, .sup.111 In, .sup.123 I, .sup.125 I or .sup.201 Tl.
There is a clinical need to be able to determine the location and/or extent of sites of focal or localized infection. In a substantial number of cases conventional methods of diagnosis (such as physical examination, x-ray, CT and ultrasonography) fail to identify such sites (e.g., an abscess). In some cases, biopsy may be resorted to, but is preferably avoided at least until it is necessary in order to identify the pathogen responsible for an abscess at a known location. Identifying the site of such "occult" infection is important because rapid localization of the problem is critical to effective therapeutic intervention.
An abscess may be caused by any one of many possible pathogens, so that a radiotracer specific for a particular pathogen would have limited scope. On the other hand, infection is almost invariably accompanied by inflammation, which is a general response of the body to tissue injury. Therefore, a radiotracer specific for sites of inflammation would be expected to be useful in localizing sites of infection caused by any pathogen.
One of the main phenomena associated with inflammation is the localization of leukocytes (white blood cells), including macrophages, monocytes and neutrophils, at the site of inflammation. A radiotracer specific for leukocytes would be useful in detecting leukocytes at the site of a localized infection.
Currently approved nuclear medicine procedures for imaging sites of infection use either indium-111 labeled leukocytes (.sup.111 In-WBC) (see, e.g. Peters, 1992, J. Nucl. Med. 33: 65-67) or gallium-67 (.sup.67 Ga) citrate (see, e.g. Ebright et al., 1982, Arch. Int. Med. 142: 246-254).
A major disadvantage of using .sup.111 In-labeled WBCs is that the preparation of the radiotracer requires sterile removal of autologous blood, sterile isolation of the leukocytes from the blood, sterile labeling of the leukocytes using conditions that do not damage the cells (since damaged WBC are taken up by the reticuloendothelial system when re-injected) and return (re-injection) of the (now labeled) leukocytes to the patient. Furthermore, a delay of 12 to 48 hours between injection and imaging may be required for optimal images. While .sup.99m Tc labeled leukocytes have been used to shorten this delay period (see, e.g. Vorne et al., 1989, J. Nucl. Med. 30: 1332-1336), ex-corporeal labeling is still required. A preferred radiotracer would be one that does not require removal and manipulation of autologous blood components.
.sup.67 Ga-citrate can be administered by intravenous injection. However, this compound is not specific for sites of infection or inflammation. Moreover, a delay of up to 72 hours is often required between injection of the radiotracer and imaging. In addition, the .gamma.-(gamma) emission energies of .sup.67 Ga are not well suited to conventional gamma cameras.
Radiolabeled monoclonal and polyclonal antibodies raised against human leukocytes (including monocytes, neutrophils, granulocytes and others) have been developed. .sup.99m Tc labeled antigranulocyte monoclonal antibodies (see, e.g. Lind et al., 1990, J. Nucl. Med. 31: 417-473) and .sup.111 In-labeled non-specific human immunoglobulin (see, e.g. LaMuraglia et al., 1989, J. Vasc. Surg. 10: 20-28) have been tested for the detection of inflammation secondary to infection. .sup.111 In-labeled IgG shares the disadvantages of .sup.111 In-labeled WBC, in that 24-48 hours are required between injection and optimal imaging. In addition, antibodies are difficult to produce and are associated with safety concerns regarding potential contamination with biological pathogens (e.g. retroviruses).
In addition, the effective treatment of cancer by surgery or radiation therapy requires knowledge of the localization and extent of the disease. A means of monitoring the progression/regression of tumors following or during any form of therapy is also highly desirable. Advances in high-resolution imaging modalities such as CT and MRI allow the detection of many neoplasms. However certain tumors and their metastases are small and difficult to localize by these methods. Nuclear medicine offers a potentially more sensitive alternative. A radiotracer that selectively binds to or localizes to any and all cancerous tissue, sufficiently to allow easy external detection, might be considered to be the ultimate goal of radiodiagnostic oncology.
Also, despite remarkable advances in cardiology, coronary artery disease remains the leading cause of death in the U.S. The final event in this disease is usually fatal myocardial infarction caused by occlusive thrombosis of one or more coronary arteries usually at the site of a complicated atherosclerotic plaque. Therefore a means, preferably non-invasive, of determining the localization and/or extent of atherosclerotic plaque is highly desirable as an aid to selecting appropriate patient management. One of the most notable characteristics of atherosclerotic plaque is the accumulation of foam cells which are lipid-laden macrophages.
.beta.-Glucans are oligoglucosides, which comprise 1,3 and 1,6 linked .beta.-D-glucose residues, originally discovered as components of yeast and fungal cell walls (Bartnicki-Garcia in Ann Rev Microbiol. 1968, 22, 87). Originally obtained in an insoluble form, .beta.-glucans have since been obtained as soluble, low molecular weight oligomers (Janusz, Austen and Czop, J. Immunol. (1989), 142, (959-965). They have been shown to be active in enhancing the host defense mechanisms of mammals by activating the alternative complement pathway through their specific binding to receptors (called .beta.-glucan receptors) found on the cell-surfaces of monocytes, macrophages and neutrophils (Czop and Kay, J. Exp. Med. (1991), 173, 1511-1520, Czop et al, Biochemistry of the Acute Allergic Reactions: Fifth International Symposium. (1989), 287-296 and J. K. Czop, Pathol. Immunopathol. Res (1986), 5, 286-296, Czop and Austen, J. Immunol. (1985), 134, 2588-2593). The in vivo administration of particulate .beta.-glucans has been shown to provide protection from many pathogens including bacteria, viruses and fungi as well as reducing tumor growth (Czop et al, Biochemistry of the Acute Allergic Reactions: Fifth International Symposium. 1989, 287-296). The smallest active .beta.-glucan reported so far is a heptaglucoside (Janusz et al, J Immunol 1989, 142, 959. Onderdonk and co-workers (Infection and Immunity, 1992, 60, 1642-1647) describe the antiinfective properties of this small .beta.-glucan. The .beta.-glucans have also been shown to exhibit an anti-tumor growth effect, believed to occur by increasing the number of macrophages localizing to tumors (Di Luzio in Pathophysiology of the Reticuloendothelial System (Eds Altruo and Saba), Raven Press, NY, pp209-224).
Czop and Janusz, U.S. Pat. No. 5,057,503 (1991), claim a heptaglucoside capable of reacting with .beta.-glucan receptors, their isolation and their therapeutic use.
Jamas et al, PCT/US90/03440 claim .beta.-glucans as drug delivery vehicles and as adjuvants.
Jamas et al, PCT/US90/05022 claim a method of activating the immune system by administering .beta.-glucans.
Jamas et al, PCT/US90/05041 claim a method of producing a soluble .beta.-glucan.
Methods for preparing radiolabel-binding moieties and of labeling them with .sup.99m Tc are disclosed in co-pending U.S. patent applications Ser. No. 07/653,012, now abandoned, which issued as U.S. Pat. No. 5,654,272; Ser. No. 07/757,470, now U.S. Pat. No. 5,225,180; Ser. No. 07/807,062, now U.S. Pat. No. 5,443,815; Ser. No. 07/851,074, now abandoned, which issued as U.S. Pat. No. 5,711,931; Ser. No. 07/871,282, a divisional of which issued as U.S. Pat. No. 5,720,934; Ser. No. 07/886,752, now abandoned, a continuation of which has been allowed as U.S. Ser. Nos. 08/273,274; 07/893,981, now U.S. Pat. No. 5,508,020; Ser. Nos. 07/955,466; 07/977,628, now U.S. Pat. No. 5,405,597; Ser. No. 08/019,525, now U.S. Pat. No. 5,552,525; Ser. No. 08/044,825, now abandoned, which issued as U.S. Pat. No. 5645,815; and Ser. No. 08/073,577, now U.S. Pat. No. 5,561,220; and PCT International Applications PCT/US92/00757, PCT/US92/10716, PCT/US93/02320, PCT/US93/03687, PCT/US93/04794, and PCT/US93/06029, which are hereby incorporated by reference.